The present invention relates generally to a method of treating human and animal disease utilizing proteolytic enzymes of plant or microbial origin (xe2x80x9cproteasesxe2x80x9d or xe2x80x9cproteinasesxe2x80x9d) and, more particularly, to a method of modifying the development or manifestation of conditions affected by the action of biologically-active molecules which may be specifically bound or stimulated by activated alpha-2-macroglobulins. Such biologically-active molecules include, but are not limited to, cytokines and other signaling molecules, such as tumor necrosis factor-alpha (TNF-xcex1), leptin, beta-amyloid (xcex2A), and transforming growth factors (TGF).
In many diseases and injuries there is a marked increase in proinflammatory cytokines in the blood. The increased cytokines are believed to contribute to the pathology of the condition. Infection, cancer and tissue injury trigger the production of cytokines. These hormone-like peptides can enter the bloodstream to alter the physiology of distant tissues, or they may behave as paracrine mediators that act only locally. In some disease or injury states, cytokines are beneficial to the host but, in others, cytokines cause the most striking manifestations of the disease (e.g., shock, tissue injury and weight loss). K. J. Tracey, et al., Tumor Necrosis Factor: A Pleiotropic Cytokine and Therapeutic Target, Ann. Rev. Med. 45: 491-503 (1994). Proteases and cytokines are intimately interrelated in the body in that cytokines are involved in regulating the production of proteases (Westermarck, J. and Kahari, V. M., Regulation of Matrix Metalloproteinase Expression in Tumor Invasion, Faseb J. 13: 781-92 (1999)), and proteases are frequently involved in the liberation of soluble cytokines. Excess circulating proteases also play an important role in the manifestation of disease or injury.
Normal human blood serum contains significant quantities of anti-proteinases, such as alpha-2-macrolobulin (xcex12M), which function to inhibit excess activity of proteases in vivo. xcex12M has a high molecular weight (Mrhuman=725,000), and is composed of two noncovalently bonded pairs of identical subunits joined by disulfide bonds. Feldman et. al., Model of xcex12-Macroglobulin Structure and Function, Proc. Nat""l. Acad. Sci. USA, 82: 5700-5704 (September 1985). The major source of plasma xcex12M is the liver (hepatocytes), although other cells including macrophages synthesize and secrete xcex12M. This may explain the presence of xcex12M in interstitial sites and malignant tissues. LaMarre et al., Cytokine Binding and Clearance Properties of Proteinase-Activated xcex12-Macroglobulins, Lab. Investigation 65(1):3-14 (1991). xcex12M inhibits proteinases of all four (4) catalytic classes. Barrett et al., The Interaction of xcex12-Macroglobulin With Proteinases: Characteristics and Specificity of the Reaction, and a Hypothesis Concerning its Molecular Mechanism, Biochem. J. 133: 709-724 (1973); Harpel et al., Studies on Human Plasma xcex12-Macroglobulin-Enzyme Interactions, J. Exp. Med. 138: 508-521 (1973); Salveson et al., Covalent Binding of Proteases in Their Reaction With xcex12-Macroglobulin, Biochem. J. 187: 695-701 (1980). Proteinases inhibited by xcex12M include the coagulation proteinases thrombin and Factor Xa, the fibrinolytic enzymes urokinase-type and tissue-type plasminogen activators as well as plasmin, kallikrein of the contact system, the neutrophilic proteinases elastase, cathepsin G, and collagenase, and several bacterial proteinases. DeBoer et al., Alpha-2-Macroglobulin Functions as an Inhibitor of Fibrinolytic, Clotting, and Neutrophilic Proteinases in Sepsis: Studies Using a Baboon Model, Infection and Immunity 61(12): 5035-5043 (1993).
Because of the biological importance of activated xcex12M (xcex12M*) scavenging of proteinases, several laboratories have attempted to elucidate the structure and function of xcex12M. The trap mechanism and structure identified by Feldman et al. has been generally accepted. According to the xe2x80x9ctrapxe2x80x9d hypothesis, proteinases cleave specific peptide bonds within the xe2x80x9cbaitxe2x80x9d amino acids sequence of the xcex12M subunits and become entrapped within the interior of the xcex12M as a result of its conformational change. Feldman et. al., supra at 5701. Methylamine mimics the proteinase-induced conformational changes by reacting with the xcex12M thiol ester bonds, and has therefore been commonly used in studies of xcex12M*.
A substantial body of research has established that xcex12M* complexes acquire a high affinity for binding proinflammatory cytokines, resulting in their removal from the body along with the protease bound in the xcex12M* complex. Activated xcex12M has been shown to play a pivotal role in regulating inflammatory and homeostatic mechanisms of disease and injury by binding major mediators such as tumor necrosis factor-alpha (TNF-xcex1), transforming growth factor-xcex21 (TGF-xcex21), transforming growth factor-xcex22 (TGF-xcex22), basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), nerve growth factor (NGF), interleukin-1xcex2(IL- 1xcex2) and interleukin-6 (IL-6). Wolleberg et.al., Binding of Tumor Necrosis Factor Alpha to Activated Forms of Human Plasma Alpha2Macroglobulin, Amer. J. Path., 138 (2): 265-272 (1991). Recent research suggests that various proinflammatory cytokines, and especially TNF-xcex1, play a major role in rheumatoid arthritis. M. Feldmann, et at., Role of Cytokines in Rheumatoid Arthritis, Ann. Rev. Immun., 14: 397-440 (1996).
One of the hallmarks of most disease processes is acute inflammation, which features the release of neutrophil-derived oxidants. It has recently been shown that xe2x80x9coxidation serves as a switch mechanism that down-regulates the progression of acute inflammation by sequestering TNF-alpha, IL-2, and IL-6, while up-regulating the development of tissue repair processes by releasing bFGF, beta-NGF, PDGF, and TGF-beta from binding to alpha2M.xe2x80x9d Wu, Patel and Pizzo, Oxidized Alpha2-Macroglobulin (alpha2M) Differentially Regulates Receptor Binding by Cytokines/Growth Factors: Iimplications for Tissue Injury and Repair Mechanisms in Inflammation. J. Immunol. 161: 4356-65 (1998). The conformationally modified or xe2x80x9cactivatedxe2x80x9d xcex12M is rapidly cleared from the circulation as it more readily binds to specific cell-surface receptors on hepatocytes, macrophages, and fibroblasts and undergoes receptor-mediated endocytosis. These cellular receptors rapidly clear xcex12M-protease and xcex12M-methylamine complexes from the systemic circulation, primarily in the liver. Native xcex12M, on the other hand, is not receptor recognized and has a prolonged half-life in circulation.
Activated xcex12M is believed to also play a role in the mediation and regulation of leptin. Leptin is a 16kDa polypeptide consisting of 167 amino acids that is expressed and secreted from adipose tissue under the control of the xe2x80x9cobese gene.xe2x80x9d Zhang Y, et al., Positional Cloning of the Mouse Obese Gene and its Human Homologue, Nature, 372:425-432 (1994). Murine studies indicate that leptin acts on the CNS to regulate body weight through the control of appetite and energy expenditure. Pelleymounter M A., et al., Effects of the Obese Gene Product on Weight Regulation in ob/ob Mice, Science, 269:540-43 (1995). Leptin has also been shown to affect sympathetic nerve activity, insulin resistance, and renal sodium excretion. Haynes W G, et al., Cardiovascular Consequences of Obesity: Role ofLeptin, Clin. Exp. Pharm. Physiol., 25:65-69 (1998). Obesity is associated with increased activity of the sympathetic nervous system and, therefore, there appears to be a causal link between excess leptin and other systemic, and especially cardiovascular consequences of obesity. See id.
There are a variety of undesirable consequences of obesity, including insulin resistance, dyslipoproteinemia, and hypercoagulability, all of which are probably due to increases in circulating TNF-xcex1 and leptin. Halle M. et al., Importance of TNF-alpha and Leptin in Obesity and Insulin Resistance: A Hypothesis on the Impact of Physical Exercise, Exerc. Immunol. Rev. 4:77-94 (1998). Activated xcex12M has been identified as a leptin binding factor in human plasma. Thus, the binding of leptin to activated xcex12M and its rapid clearance by the xcex12M receptor can significantly influence the bioavailability of leptin. Birkenmeier G., et al., Human Leptin Forms Complexes with xcex12-Macroglobulin Which are Recognized by the xcex12-Macroglobulin Receptor/Low Density Lipoprotein Receptor-Related Protein, Eur. J. Endocrin., 139:224-230 (1998).
TNF-xcex1 is thought to be a modulator of gene expression in adipocytes and is implicated in the development of insulin resistance and obesity. Thus, the clearance of TNF-xcex1 by activated xcex12Ms also appears desirable. Fernandez-Real J M, et al., The TNF-alpha Gene NCO I Polymorphism Influences the relationship Among Insulin resistance, Percent Body Fat, and Increased Serum Leptin Levels, Diabetes 46(9):1468-72 (1997). The Fernandez-Real group found that increasing transcription of TNF-xcex1 using a polymorphism on the TNF-xcex1 gene increased serum leptin concentrations in a sample of human subjects. Similarly, diet-induced weight loss reduced TNF-xcex1 expression and serum leptin levels and improved insulin sensitivity and lipid metabolism. Halle M. et al., Importance of TNF-alpha and Leptin in Obesity and Insulin Resistance: a Hypothesis on the Impact of Physical Exercise, Exerc. Immunol. Rev. 4:77-94 (1998). A composition that activates xcex12Ms can also be useful for the treatment of non-insulin dependent diabetes and insulin-resistance effect (See Morimoto et al., Life Science, 61:795-803 (1997)), Crohn""s disease (See U.S. Pat. No. 5,656,272) and cachexia (See Tisdale, Wasting and Cancer, J. Nutrition, 129 (1S Suppl.): 243S-246S.
Activated xcex12Ms also affect the clearance of beta-amyloid (xcex2A). Studies show that increased deposition and aggregation of xcex2A is one of the principal neuropathological features of Alzheimer""s disease (AD). Selkoe D J, Cell Biology of the Amyloid xcex2-Protein Precursor and the Mechanism of Alzheimer""s Disease, Ann. Rev. Cell Biol., 10:373-403 (1994). Amyloid deposits comprise a 39-43 amino acid peptide(s), which is a proteolytic processing product of the amyloid precursor protein (APP), that is expressed by most, if not all, cells. Once formed, the xcex2A peptide oligomerizes and aggregates into insoluble fibrils that are directly toxic to neurons. Pike C. J., et al., In- Vitro Aging of xcex2-Amyloid Protein Causes Peptide Aggregation and Neurotoxicity, Brain Res., 563:311-314 (1991). It has been found that the circulating or brain concentration of xcex2A in patients with AD is greater than normal patients, and it is believed that factors contributing to xcex2A catabolism and/or clearance of xcex2A may contribute to either diffuse (preamyloid) or neuritic (senile) plaques in the brain. Van Gool D., et al., xcex12-Macroglobulin Expression in Neuritic-Type Plaques in Patients with Alzheimer""s Disease, Neurobiol. Aging, 14:233-37 (1993).
Using radiolabeled xcex2A, Du et al. found that xcex12M binds to xcex2A with high affinity. Du Y, et al., xcex12-Macroglobulin as a xcex2-Amyloid Peptide-Binding Plasma Protein, J. Neurochem., 69:299-305 (1997). This indicates that xcex2A is cleared from the brain by conjugation with xcex12M and endocytosis of the xcex2A/xcex12M complex by low-density lipoprotein receptor-related protein (LRP). Investigations found that xcex12M in AD brain were localized to neuritic plaques and that xcex12M receptors, or LRP, were concentrated in brain areas affected by AD. Strauss S., Detection of Interleukin-6 and xcex12-Macroglobulin Immunoreactivity in Cortex and Hippocampus of Alzheimer""s Disease, Lab. Invest., 66:223-230 (1992).
It has also been discovered that activated xcex12M directly stimulates macrophages. See Misra and Pizzo, Ligation Of The Alpha2 Signaling Receptor With Receptor-Recognized Forms Of Alpha2-Macroglobulin Initiates Protein And DNA Synthesis In Macrophages: The Effect Of Intracellular Calcium. Biochim. Biophys. Acta.,1401:121-8 (1998). Such macrophage stimulation can have beneficial effects such as fighting bacterial infection.
Despite the growing body of research implicating xcex12M* as a mediator of various diseases, pharmacotherapy has failed to address modulating xcex12M to effect treatment of the diseases. In particular, the prior art has failed to focus on modulating xcex12Ms with agents that are effective, well tolerated by most patients and economical to use. Most current therapy is directed to altering the function of the cytokines themselves. For example, EMBREL, the commercial product based on Le et al. U.S. Pat. No. 5,698,195, Methods of Treating Rheumatoid Arthritis Using Chimeric Anti-TNF Antibodies issued Dec. 16, 1997, teaches the use of anti-TNF antibodies specific for human tumor necrosis factor-alpha for the treatment of rheumatoid arthritis. Such treatment, however, has the disadvantages of being expensive at approximately $220.00 per week of therapy and is administered by injection and therefore carries the associated risks.
Additionally, Mynott U.S. Pat. No. 5,824,305 focuses on a different mechanism and teaches a method of treating diseases mediated by cyclic nucleotide pathways with purified stem bromelain protease.
Activated xcex12Ms play a key role in influencing the availability and activity of various peptides to specific cells and, therefore, influencing cellular physiology. Consequently, there is a definite need in the art of mammalian therapeutics for a pharmacological agent that increases the activated or xe2x80x9cfastxe2x80x9d form of xcex12M to mediate the effects of the above-mentioned cytokines and other signaling molecules, has a low side effect profile, and is economical to use.
In accordance with the present invention, it has now been discovered that certain manifestations of disease and injury may be effectively treated with a mixture of proteases of microbial and/or plant origin. Specifically, exogenous proteases are useful for increasing activated alpha-2-macroglobulin in the blood and extravascular tissue. Such proteases are preferably administered orally, either singly or in combination with synergistic ingredients, in the form of capsules (hard and soft), tablets (film coated, enteric coated or uncoated), powder or granules (film coated, enteric coated or uncoated) or liquid (solution or suspension) in an amount to produce the desired pharmacological effect, namely objective improvement of the condition of treated patients by positively influencing the cellular physiology.
The protease may be any pharmaceutically acceptable protease, and preferably is of microbial and/or plant origin, given singly or in combination with vitamins, minerals, antioxidants, bioflavonoids, proanthocyanidins, herbs, herbal extracts, plant and animal concentrates, and analgesics. The microbial protease is preferably administered in a total daily dosage of at least 100,000 HUT (or equivalent biological activity). The plant protease is preferably administered in a total daily dosage of at least 50,000 PU (or equivalent biological activity).
A specific composition and method of use thereof for promoting recovery from soft tissue injury is also disclosed. The orally-administered composition contains a mixture of microbial and plant proteases, antioxidant bioflavonoids, proanthocyanidins, vitamins, minerals, plant concentrates and excipients. The composition can also include an analgesic.
It is, therefore, an object of the present invention is to treat mammalian disease or injury manifested by cytokines and other signaling molecules by administering to a mammal oral doses of protease to increase activated xcex122M*in the serum, which in turn serves as a biological response modifier for the disease or injury. The present invention can be employed to treat any disease or injury where activated xcex12M* play a role.
It is another object of the present invention to supply exogenous protease to the circulation to create a xe2x80x9cpreemptivexe2x80x9d increase in xcex12M* capable of binding proinflammatory cytokines and thereby interrupting cytokine-induced pathology.
It is also an object of the present invention to provide a protease-based composition and method for decreasing the time required for healing of soft tissue injuries resulting from accidents, sports injuries, various surgical procedures and the like.
It is yet another object of the present invention to treat disease or injury with a composition that is easy to administer, economical and well-tolerated by patients.